At least three components of human epidermal cells participate in the formation of cross-linked envelopes: envelope precursor protein, which represents the structural subunit of envelopes; plasma membrane, where subunit assembly takes place; and transglutaminase, which stabilizes the assembled structure with epsilon- (gamma-glutamyl)-lysine cross-links. The first part of the work involves purification of transglutaminase from the cultured cells and characterization of the chromatographically distinct species that are detectable. The enzymes will be compared in physical parameters and in specificities for synthetic and natural substrates. In addition, the envelope precursor protein will be characterized for measurement of its glutamine to glutamic acid ratio, for clustering of Glx and hydrophobic residues, and for estimation of the number of Gln residues available for transglutaminase modification. Further, the propensity of the soluble precursor to form homopolymers will be examined in the presence of transglutaminase and, in some experiments, with other proteins serving as amine donors. The ability of synthetic glutamine-containing polypeptides to bind transglutaminase and to be cross-linked will be considered. The second aspect of this proposal involves reconstitution of envelopes in vitro and a search for other possible components participating in assembly. The binding of envelope subunit protein to keratinocyte microsomes will be investigated. For comparison, binding to fibroblast microsomes, red cell vesicles, and synthetic liposomes will also be studied. Direct approaches to identifying other proteins involved in envelope assembly will be tried, including 1) testing cell extracts for antigens (other than precursor protein) binding to antiserum directed against purified envelopes; 2) release of minimally attached proteins for isolated envelopes by transglutaminase-mediated reversal of cross-linking; and 3) cross-linking assembled envelopes with agents containing labile bonds, permitting recovery of proteins proximal to envelope precursor. Finally, keratinocyte lines recently derived from human tumors will be surveyed for alterations in envelope formation in culture. Those lines found defective will be studied in detail.